PROPOSAL SUMMARY/ABSTRACT Streptococcus pneumoniae kills more people in the U.S. than all other vaccine-preventable diseases combined and is estimated to cause over 1.6 million deaths/year worldwide. The native pneumococcal vaccine currently used in adults consists of capsular polysaccharides (PPS) derived from 23 different serotypes and therefore provides broad coverage against invasive disease with an estimated efficacy of ~65%. Nonetheless, protection eventually wanes as antibody titers diminish, typically by 5-10 years post-immunization. Unfortunately, PPS immunization elicits mainly IgM and PPS boosting does not induce recall responses. This is typical of most polysaccharide (Ps) antigens. This is a significant concern given the potential for diminished protection against pneumococcal infections. PPS-protein conjugate vaccines effectively boost in children, but in adults, do not yield superior titers to native PPS or effectively boost. This, along with the high cost and limited serotype coverage of conjugate vaccines, suggests that alternative strategies are needed to enhance PPS vaccine efficacy. To improve native Ps vaccines, it is imperative to identify adjuvants that can supply a second stimulatory, or ?danger?, signal to Ps-specific B cells. Our data indicate that MyD88-dependent signals supplied through B cell-expressed toll-like receptor 4 (TLR4) significantly increase Ab responses to Ps antigens and support Ps boosting in mice. However, TLR4 expression is low to negative on human B cells. Therefore, the goal of this project is to identify TLR that activate MyD88-dependent signaling on Ps-specific human B cells so that optimal adjuvants can be designed for human Ps vaccines. Aim 1, we will specifically examine the TLR expression patterns of Ps-specific B cells in humans, during the course of an immune response to Ps using both in vitro and in vivo approaches to analyze human and non-human primate (NHP) B cells. We will then determine optimal TLR agonists or combinations of agonists that promote proliferation, survival, and/or increased differentiation of Ps-specific B cells in an in vitro Ps-specific B cell activation assay. In Aim 2, we will use humanized and primatized mice to test lead TLR agonists or agonist combinations as effective adjuvants for different Ps antigens, including distinct PPS. Finally, we will test the extent to which these TLR agonist- elicited Ps-specific antibodies provide protection against pneumococcal challenge. Our results are expected to have a significant impact on the future design of more affordable and efficacious Ps-based vaccines.